The functionality and translatability of neocartilage constructs are improved with the combination of fluid-induced shear stress and bioactive factors.

Neocartilage tissue engineering aims to address the shortcomings of current clinical treatments for articular cartilage indications. However, advancement is required toward neocartilage functionality (mechanical and biochemical properties) and translatability (construct size, gross morphology, passage number, cell source, and cell type). Using fluid-induced shear (FIS) stress, a potent mechanical stimulus, over four phases, this work investigates FIS stress' efficacy toward creating large neocartilage derived from highly passaged minipig costal chondrocytes, a species relevant to the preclinical regulatory process. In Phase I, FIS stress application timing was investigated in bovine articular chondrocytes and found to improve the aggregate modulus of neocartilage by 151% over unstimulated controls when stimulated during the maturation stage. In Phase II, FIS stress stimulation was translated from bovine articular chondrocytes to expanded minipig costal chondrocytes, yielding a 46% improvement in aggregate modulus over nonstimulated controls. In Phase III, bioactive factors were combined with FIS stress to improve the shear modulus by 115% over bioactive factor-only controls. The translatability of neocartilage was improved in Phase IV by utilizing highly passaged cells to form constructs more than 9-times larger in the area (11 × 17 mm), yielding an improved aggregate modulus by 134% and a flat morphology compared to free-floating, bioactive factor-only controls. Overall, this study represents a significant step toward generating mechanically robust, large constructs necessary for animal studies, and eventually, human clinical studies.

Vertical dimension in dental sleep medicine oral appliance therapy.

The objective of this pilot study was to evaluate the effect of a multidimensional approach to occlusal registration, including vertical dimension as assessed using pharyngometry, on the success of oral appliance therapy. Successful medical improvements resulting from therapy were determined by secondary polysomnographic studies. Thirty patients were enrolled in this pilot study. Their initial apnea-hypopnea index (AHI) scores ranged from 6.0 (mild obstructive sleep apnea) to 81.6 (severe obstructive sleep apnea). Occlusal registrations were taken using pharyngometer readings to establish vertical and anteroposterior (AP) positions for each patient and compared to the AP-only position in the same patient, determined using a George Gauge at 70% protrusion. All follow-up sleep tests occurred 31-45 days after the delivery of oral appliances set at the multidimensional vertical and AP positions determined by pharyngometry. No appliance titration was required. In the 26 patients who completed the study, the mean AHI before therapy was 20.7, and the mean AHI after therapy was 7.8, a 62.3% decrease. Within the limitations of this study, the pharyngometer-established occlusal position was effective in lowering AHI without the need for appliance titration procedures, which are typically required when the 70% protrusive George Gauge occlusal registration method is used. Additionally, the position determined with the 70% George Gauge was, on average, 5.0 mm more protrusive than the pharyngometer registration.

Facet Joints of the Spine: Structure-Function Relationships, Problems and Treatments, and the Potential for Regeneration.

The zygapophysial joint, a diarthrodial joint commonly referred to as the facet joint, plays a pivotal role in back pain, a condition that has been a leading cause of global disability since 1990. Along with the intervertebral disc, the facet joint supports spinal motion and aids in spinal stability. Highly susceptible to early development of osteoarthritis, the facet is responsible for a significant amount of pain in the low-back, mid-back, and neck regions. Current noninvasive treatments cannot offer long-term pain relief, while invasive treatments can relieve pain but fail to preserve joint functionality. This review presents an overview of the facet in terms of its anatomy, functional properties, problems, and current management strategies. Furthermore, this review introduces the potential for regeneration of the facet and particular engineering strategies that could be employed as a long-term treatment.

Tension stimulation drives tissue formation in scaffold-free systems.

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue.

Considerations for translation of tissue engineered fibrocartilage from bench to bedside.

Fibrocartilage is found in the knee meniscus, the temporomandibular joint (TMJ) disc, the pubic symphysis, the annulus fibrosus of intervertebral disc, tendons, and ligaments. These tissues are notoriously difficult to repair due to their avascularity, and limited clinical repair and replacement options exist. Tissue engineering has been proposed as a route to repair and replace fibrocartilages. Using the knee meniscus and TMJ disc as examples, this review describes how fibrocartilages can be engineered toward translation to clinical use. Presented are fibrocartilage anatomy, function, epidemiology, pathology, and current clinical treatments because they inform design criteria for tissue engineered fibrocartilages. Methods for how native tissues are characterized histomorphologically, biochemically, and mechanically to set gold standards are described. Then, provided is a review of fibrocartilage-specific tissue engineering strategies, including the selection of cell sources, scaffold or scaffold-free methods, and biochemical and mechanical stimuli. In closing, the Food and Drug Administration paradigm is discussed to inform researchers of both the guidance that exists and the questions that remain to be answered with regard to bringing a tissue engineered fibrocartilage product to the clinic.

Articular cartilage tissue engineering: the role of signaling molecules.

Effective early disease modifying options for osteoarthritis remain lacking. Tissue engineering approach to generate cartilage in vitro has emerged as a promising option for articular cartilage repair and regeneration. Signaling molecules and matrix modifying agents, derived from knowledge of cartilage development and homeostasis, have been used as biochemical stimuli toward cartilage tissue engineering and have led to improvements in the functionality of engineered cartilage. Clinical translation of neocartilage faces challenges, such as phenotypic instability of the engineered cartilage, poor integration, inflammation, and catabolic factors in the arthritic environment; these can all contribute to failure of implanted neocartilage. A comprehensive understanding of signaling molecules involved in osteoarthritis pathogenesis and their actions on engineered cartilage will be crucial. Thus, while it is important to continue deriving inspiration from cartilage development and homeostasis, it has become increasingly necessary to incorporate knowledge from osteoarthritis pathogenesis into cartilage tissue engineering.

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking.

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

TGF-ß1, GDF-5, and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells.

Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor ß1 (TGF-ß1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-ß1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-ß1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-ß1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs.

Recent Tissue Engineering Advances for the Treatment of Temporomandibular Joint Disorders.

Temporomandibular disorders (TMDs) are among the most common maxillofacial complaints and a major cause of orofacial pain. Although current treatments provide short- and long-term relief, alternative tissue engineering solutions are in great demand. Particularly, the development of strategies, providing long-term resolution of TMD to help patients regain normal function, is a high priority. An absolute prerequisite of tissue engineering is to understand normal structure and function. The current knowledge of anatomical, mechanical, and biochemical characteristics of the temporomandibular joint (TMJ) and associated tissues will be discussed, followed by a brief description of current TMD treatments. The main focus is on recent tissue engineering developments for regenerating TMJ tissue components, with or without a scaffold. The expectation for effectively managing TMD is that tissue engineering will produce biomimetic TMJ tissues that recapitulate the normal structure and function of the TMJ.

Harnessing biomechanics to develop cartilage regeneration strategies.

As this review was prepared specifically for the American Society of Mechanical Engineers H.R. Lissner Medal, it primarily discusses work toward cartilage regeneration performed in Dr. Kyriacos A. Athanasiou's laboratory over the past 25 years. The prevalence and severity of degeneration of articular cartilage, a tissue whose main function is largely biomechanical, have motivated the development of cartilage tissue engineering approaches informed by biomechanics. This article provides a review of important steps toward regeneration of articular cartilage with suitable biomechanical properties. As a first step, biomechanical and biochemical characterization studies at the tissue level were used to provide design criteria for engineering neotissues. Extending this work to the single cell and subcellular levels has helped to develop biochemical and mechanical stimuli for tissue engineering studies. This strong mechanobiological foundation guided studies on regenerating hyaline articular cartilage, the knee meniscus, and temporomandibular joint (TMJ) fibrocartilage. Initial tissue engineering efforts centered on developing biodegradable scaffolds for cartilage regeneration. After many years of studying scaffold-based cartilage engineering, scaffoldless approaches were developed to address deficiencies of scaffold-based systems, resulting in the self-assembling process. This process was further improved by employing exogenous stimuli, such as hydrostatic pressure, growth factors, and matrix-modifying and catabolic agents, both singly and in synergistic combination to enhance neocartilage functional properties. Due to the high cell needs for tissue engineering and the limited supply of native articular chondrocytes, costochondral cells are emerging as a suitable cell source. Looking forward, additional cell sources are investigated to render these technologies more translatable. For example, dermis isolated adult stem (DIAS) cells show potential as a source of chondrogenic cells. The challenging problem of enhanced integration of engineered cartilage with native cartilage is approached with both familiar and novel methods, such as lysyl oxidase (LOX). These diverse tissue engineering strategies all aim to build upon thorough biomechanical characterizations to produce functional neotissue that ultimately will help combat the pressing problem of cartilage degeneration. As our prior research is reviewed, we look to establish new pathways to comprehensively and effectively address the complex problems of musculoskeletal cartilage regeneration.

Self-organization and the self-assembling process in tissue engineering.

In recent years, the tissue engineering paradigm has shifted to include a new and growing subfield of scaffoldless techniques that generate self-organizing and self-assembling tissues. This review aims to cogently describe this relatively new research area, with special focus on applications toward clinical use and research models. Particular emphasis is placed on providing clear definitions of self-organization and the self-assembling process, as delineated from other scaffoldless techniques in tissue engineering and regenerative medicine. Significantly, during formation, self-organizing and self-assembling tissues display biological processes similar to those that occur in vivo. These processes help lead to the recapitulation of native tissue morphological structure and organization. Notably, functional properties of these engineered tissues, some of which are already in clinical trials, also approach native tissue values. This review endeavors to provide a cohesive summary of work in this field and to highlight the potential of self-organization and the self-assembling process for providing cogent solutions to currently intractable problems in tissue engineering.

Clinical translation of stem cells: insight for cartilage therapies.

The limited regenerative capacity of articular cartilage and deficiencies of current treatments have motivated the investigation of new repair technologies. In vitro cartilage generation using primary cell sources is limited by cell availability and expansion potential. Pluripotent stem cells possess the capacity for chondrocytic differentiation and extended expansion, providing a potential future solution to cell-based cartilage regeneration. However, despite successes in producing cartilage using adult and embryonic stem cells, the translation of these technologies to the clinic has been severely limited. This review discusses recent advances in stem cell-based cartilage tissue engineering and the major current limitations to clinical translation of these products. Concerns regarding appropriate animal models and studies, stem cell manufacturing, and relevant regulatory processes and guidelines will be addressed. Understanding the significant hurdles limiting the clinical use of stem cell-based cartilage may guide future developments in the fields of tissue engineering and regenerative medicine.

A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross-linking and biomechanical properties in engineered neocartilage.

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.

Topographic variations in biomechanical and biochemical properties in the ankle joint: an in vitro bovine study evaluating native and engineered cartilage.

PURPOSE: The purposes of this study were to identify differences in the biomechanical and biochemical properties among the articulating surfaces of the ankle joint and to evaluate the functional and biological properties of engineered neocartilage generated using chondrocytes from different locations in the ankle joint. METHODS: The properties of the different topographies within the ankle joint (tibial plafond, talar dome, and distal fibula) were evaluated in 28 specimens using 7 bovine ankles; the femoral condyle was used as a control. Chondrocytes from the same locations were used to form 28 neocartilage constructs by tissue engineering using an additional 7 bovine ankles. The functional properties of neocartilage were compared with native tissue values. RESULTS: Articular cartilage from the tibial plafond, distal fibula, talar dome, and femoral condyle exhibited Young modulus values of 4.8 ± 0.5 MPa, 3.9 ± 0.1 MPa, 1.7 ± 0.2 MPa, and 4.0 ± 0.5 MPa, respectively. The compressive properties of the corresponding tissues were 370 ± 22 kPa, 242 ± 18 kPa, 255 ± 26 kPa, and 274 ± 18 kPa, respectively. The tibial plafond exhibited 3-fold higher tensile properties and 2-fold higher compressive and shear moduli compared with its articulating talar dome; the same disparity was observed in neocartilage. Similar trends were detected in biochemical data for both native and engineered tissues. CONCLUSIONS: The cartilage properties of the various topographic locations within the ankle are significantly different. In particular, the opposing articulating surfaces of the ankle have significantly different biomechanical and biochemical properties. The disparity between tibial plafond and talar dome cartilage and chondrocytes warrants further evaluation in clinical studies to evaluate their exact role in the pathogenesis of ankle lesions. CLINICAL RELEVANCE: Therapeutic modalities for cartilage lesions need to consider the exact topographic source of the cells or cartilage grafts used. Furthermore, the capacity of generating neocartilage implants from location-specific chondrocytes of the ankle joint may be used in the future as a tool for the treatment of chondral lesions.

Recent advancements in cartilage tissue engineering innovation and translation.

Articular cartilage was expected to be one of the first successfully engineered tissues, but today, cartilage repair products are few and they exhibit considerable limitations. For example, of the cell-based products that are available globally, only one is marketed for non-knee indications, none are indicated for severe osteoarthritis or rheumatoid arthritis, and only one is approved for marketing in the USA. However, advances in cartilage tissue engineering might now finally lead to the development of new cartilage repair products. To understand the potential in this field, it helps to consider the current landscape of tissue-engineered products for articular cartilage repair and particularly cell-based therapies. Advances relating to cell sources, bioactive stimuli and scaffold or scaffold-free approaches should now contribute to progress in therapeutic development. Engineering for an inflammatory environment is required because of the need for implants to withstand immune challenge within joints affected by osteoarthritis or rheumatoid arthritis. Bringing additional cartilage repair products to the market will require an understanding of the translational vector for their commercialization. Advances thus far can facilitate the future translation of engineered cartilage products to benefit the millions of patients who suffer from cartilage injuries and arthritides.

Detrimental Effects of Chlorhexidine on Articular Cartilage Viability, Matrix, and Mechanics.

BACKGROUND: Chlorhexidine gluconate (CHG) solution is commonly used as an antiseptic irrigation for bacterial decontamination during orthopaedic surgery. Although the chondrotoxicity of CHG on articular cartilage has been reported, the full extent of CHG-related chondrotoxicity and its effects on the extracellular matrix and mechanical properties are unknown. PURPOSE: To investigate the in vitro effects of a single 1-minute CHG exposure on the viability, biochemical content, and mechanics of native articular cartilage explants. STUDY DESIGN: Controlled laboratory study. METHODS: Articular cartilage explants (6 per group) were harvested from femoral condyles of the porcine stifle and sectioned at tidemark. Explants were bathed in CHG solution (0.05% CHG in sterile water) at varying concentrations (0% control, 0.01% CHG, and 0.05% CHG) for 1 minute, followed by complete phosphate-buffered saline wash and culture in chondrogenic medium. At 7 days after CHG exposure, cell viability, matrix content (collagen and glycosaminoglycan [GAG]), and compressive mechanical properties (creep indentation testing) were assessed. RESULTS: One-minute CHG exposure was chondrotoxic to explants, with both 0.05% CHG (2.6% ± 4.1%) and 0.01% CHG (76.3% ± 8.6%) causing a decrease in chondrocyte viability compared with controls (97.5% ± 0.6%; P < .001 for both). CHG exposure at either concentration had no significant effect on collagen content, while 0.05% CHG exposure led to a significant decrease in mean GAG per wet weight compared with the control group (2.6% ± 1.7% vs 5.2% ± 1.9%; P = .029). There was a corresponding weakening of mechanical properties in explants treated with 0.05% CHG compared with controls, with decreases in mean aggregate modulus (177.8 ± 90.1 kPa vs 280.8 ± 19.8 kPa; P < .029) and shear modulus (102.6 ± 56.5 kPa vs 167.9 ± 16.2 kPa; P < .020). CONCLUSION: One-minute exposure to CHG for articular cartilage explants led to dose-dependent decreases in chondrocyte viability, GAG content, and compressive mechanical properties. This raises concern for the risk of mechanical failure of the cartilage tissue after CHG exposure. CLINICAL RELEVANCE: Clinicians should be judicious regarding the use of CHG irrigation at these concentrations in the presence of native articular cartilage.

Biomechanical, biochemical, and histological characterization of sacroiliac joint cartilage in the Yucatan minipig.

Although the sacroiliac (SI) joint can be a source of lower back and buttock pain, no comprehensive characterization studies on SI cartilage have been conducted. Using the minipig as a large animal model, this study conducted the first biomechanical, biochemical, and histological characterization of SI joint cartilage. Because previous literature has reported that sacral cartilage and iliac cartilage within the SI joint are histologically distinct, concomitantly it was expected that functional properties of the sacral cartilage would differ from those of the iliac cartilage. Creep indentation, uniaxial tension, biochemical, and histological analyses were conducted on the sacral and iliac cartilage of skeletally mature female Yucatan minipigs (n = 6-8 for all quantitative tests). Concurring with prior literature, the iliac cartilage appeared to be more fibrous than the sacral cartilage. Glycosaminoglycan content was 2.2 times higher in the sacral cartilage. The aggregate modulus of the sacral cartilage was 133 ± 62 kPa, significantly higher than iliac cartilage, which only had an aggregate modulus of 51 ± 61 kPa. Tensile testing was conducted in both cranial-caudal and ventral-dorsal axes, and Young's modulus values ranged from 2.5 ± 1.5 MPa to 13.6 ± 1.5 MPa, depending on anatomical structure (i.e., sacral vs. iliac) and orientation of the tensile test. The Young's modulus of sacral cartilage was 5.5 times higher in the cranial-caudal axis and 2.0 times higher in the ventral-dorsal axis than the iliac cartilage. The results indicate that the sacral and iliac cartilages are functionally distinct from each other. Understanding the distinct differences between sacral and iliac cartilage provides insight into the structure and function of the SI joint, which may inform future research aimed at repairing SI joint cartilage.

A cell bank paradigm for preclinical evaluation of an analogous cellular product for an allogeneic cell therapy.

Toward the translation of allogeneic cell therapy products, cell banks are needed not only to manufacture the final human product but also during the preclinical evaluation of an animal-based analogous cellular product (ACP). These cell banks need to be established at both the master cell bank (MCB) level and the working cell bank (WCB) level. Inasmuch as most of the development of cell therapy products is at academic centers, it is imperative that academic researchers understand how to establish MCBs and WCBs within an academic environment. To illustrate this process, using articular cartilage as the model, a cell bank for an ACP was developed (MCBs at passage 2, WCBs at passage 5) to produce self-assembled neocartilage for preclinical evaluation (constructs at passage 7). The cell bank system is estimated to be able to produce between 160 000 and 400 000 constructs for each of the six MCBs. Overall, the ACP cell bank yielded constructs that are analogous to the intended human product, which is critical toward conducting preclinical evaluations of the ACP for inclusion in an Investigational New Drug application to the FDA.

Biomechanics-driven chondrogenesis: from embryo to adult.

Biomechanics plays a pivotal role in articular cartilage development, pathophysiology, and regeneration. During embryogenesis and cartilage maturation, mechanical stimuli promote chondrogenesis and limb formation. Mechanical loading, which has been characterized using computer modeling and in vivo studies, is crucial for maintaining the phenotype of cartilage. However, excessive or insufficient loading has deleterious effects and promotes the onset of cartilage degeneration. Informed by the prominent role of biomechanics, mechanical stimuli have been harnessed to enhance redifferentiation of chondrocytes and chondroinduction of other cell types, thus providing new chondrocyte cell sources. Biomechanical stimuli, such as hydrostatic pressure or compression, have been used to enhance the functional properties of neocartilage. By identifying pathways involved in mechanical stimulation, chemical equivalents that mimic mechanical signaling are beginning to offer exciting new methods for improving neocartilage. Harnessing biomechanics to improve differentiation, maintenance, and regeneration is emerging as pivotal toward producing functional neocartilage that could eventually be used to treat cartilage degeneration.

Characterization of the Temporomandibular Joint Disc Complex in the Yucatan Minipig.

The temporomandibular joint (TMJ) disc complex (i.e., the TMJ disc and its six attachments) is crucial to everyday functions such as mastication and speaking. The TMJ can be afflicted by many conditions, including disc displacement and defects. Pathologies of the TMJ disc complex most commonly present first as anterior disc displacement, which the field hypothesizes may implicate the two posterior attachments. As a result of anterior disc displacement, defects may develop in the lateral disc complex. Tissue engineering is poised to improve treatment paradigms for these indications of the TMJ disc complex by engineering biomimetic implants, but, first, gold-standard design criteria for such implants should be established through characterization studies. This study's objective was to characterize the structural, mechanical, biochemical, and crosslinking differences among the two posterior attachments and the lateral disc in the Yucatan minipig, a well-accepted TMJ animal model. In tension, it was found that the posterior inferior attachment (PIA) was significantly stiffer and stronger by 2.13 and 2.30 times, respectively, than the posterior superior attachment (PSA). It was found that collagen in both attachments was primarily aligned mediolaterally; however, the lateral disc was much more aligned and anisotropic than either attachment. Among the three locations, the PSA exhibited the greatest degree of heterogeneity and highest proportion of fat vacuoles. The PIA and lateral disc were 1.93 and 1.91 times more collagenous, respectively, by dry weight (DW) than the PSA. The PIA also exhibited 1.78 times higher crosslinking per DW than the PSA. Glycosaminoglycan per DW was significantly higher in the lateral disc by 1.48 and 5.39 times than the PIA and PSA, respectively. Together, these results establish design criteria for tissue-engineering of the TMJ disc complex and indicate that the attachments are less fibrocartilaginous than the disc, while still significantly contributing to the mechanical stability of the TMJ disc complex during articulation. These results also support the biomechanical function of the PIA and PSA, suggesting that the stiffer PIA anchors the disc to the mandibular condyle during articulation, while the softer PSA serves to allow translation over the articular eminence. Impact Statement Characterization of the temporomandibular joint (TMJ) disc complex (i.e., the disc and its attachments) has important implications for those aiming to tissue-engineer functional replacements and can help elucidate its biomechanical function. For example, the findings shown here suggest that the stiffer posterior inferior attachment anchors the disc during articulation, while the softer posterior superior attachment allows translation over the articular eminence.